scholarly journals High-Throughput Method for Rapid Induction of Prophages from Lysogens and Its Application in the Study of Shiga Toxin-Encoding Escherichia coli Strains

2010 ◽  
Vol 76 (7) ◽  
pp. 2360-2365 ◽  
Author(s):  
James E. McDonald ◽  
Darren L. Smith ◽  
Paul C. M. Fogg ◽  
Alan J. McCarthy ◽  
Heather E. Allison
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Shiga Toxin ◽  
Large Scale ◽  
Plaque Assay ◽  
Bacterial Strains ◽  
Gene Markers ◽  
Rapid Induction ◽  
Bacterial Indicator ◽  
Culture Liquor

ABSTRACT A high-throughput 96-well plate-based method for the rapid induction of endogenous prophages from individual bacterial strains was developed. The detection of endogenous prophages was achieved by the filtration of the culture liquor following norfloxacin induction and subsequent PCRs targeting bacteriophage-carried gene markers. The induction method was tested on 188 putative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains and demonstrated the ability to detect both lambdoid and stx-carrying bacteriophages in strains for which plaques were not observed via plaque assay. Lambdoid bacteriophages were detected in 37% of the induced phage preparations via amplification of the Q gene, and Stx1- and Stx2-encoding phages were detected in 2 and 14% of the strains, respectively. The method therefore provided greater sensitivity for the detection of Stx and other lambdoid bacteriophage populations carried by STEC strains than that for the established method of plaque assay using bacterial indicator strains, enabling, for the first time, large-scale bacteriophage population and diversity studies.

Enterohemorrhagic Escherichia coli Colony Check Assay for the Identification of Serogroups O26, O45, O103, O111, O121, O145, and O157 Colonies Isolated on Plating Media

2014 ◽  
Vol 77 (7) ◽  
pp. 1212-1218 ◽  
Author(s):  
BURTON BLAIS ◽  
MYLÈNE DESCHÊNES ◽  
GEORGE HUSZCZYNSKI ◽  
MARTINE GAUTHIER
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
High Throughput Screening ◽  
Enterohemorrhagic Escherichia Coli ◽  
Test Results ◽  
Bacterial Strains ◽  
Enrichment Broth ◽  
Substrate Solution ◽  
E Coli ◽  
Assay Performance

A simple immunoenzymatic enterohemorrhagic Escherichia coli (EHEC) colony check (ECC) assay was developed for the presumptive identification of priority EHEC colonies isolated on plating media from enrichment broth cultures of foods. With this approach, lipopolysaccharide extracted from a colony is spotted on the grid of a polymyxin-coated polyester cloth strip, and bound E. coli serogroup O26, O45, O103, O111, O121, O145, and O157 antigens are subsequently detected by sequential reactions with a pool of commercially available peroxidase-conjugated goat antibodies and tetramethylbenzidine substrate solution. Each strip can accommodate up to 15 colonies, and test results are available within 30 min. Assay performance was verified using colonies from a total of 73 target EHEC isolates covering the range of designated priority serogroups (all of which were reactive), 41 nontarget E. coli isolates including several nontarget Shiga toxin–producing E. coli serogroups (all unreactive), and 33 non–E. coli strains (all unreactive except two bacterial strains possessing O-antigenic structures in common with those of the priority EHEC). The ECC assay was reactive with target colonies grown on several types of selective and nonselective plating media designed for their cultivation. These results support the use of the ECC assay for high-throughput screening of colonies isolated on plating media for detecting priority EHEC strains in foods.

scholarly journals Melanin-Based High-Throughput Screen for l-Tyrosine Production in Escherichia coli

2007 ◽  
Vol 74 (4) ◽  
pp. 1190-1197 ◽  
Author(s):  
Christine Nicole S. Santos ◽  
Gregory Stephanopoulos
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Combinatorial Libraries ◽  
Selection Strategy ◽  
High Throughput Screen ◽  
Bacterial Strains ◽  
Gene Encoding ◽  
Strong Linear Correlation ◽  
Low Levels ◽  
Tyrosine Content

ABSTRACT We present the development of a simple, high-throughput screen for identifying bacterial strains capable of l-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link l-tyrosine production in Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular l-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for l-tyrosine-overproducing strains.

scholarly journals Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

2016 ◽  
Vol 54 (11) ◽  
pp. 2711-2715
Author(s):  
Agatha N. Jassem ◽  
Frank Y. Chou ◽  
Cathevine Yang ◽  
Matthew A. Croxen ◽  
Katarina D. M. Pintar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Shiga Toxin ◽  
Large Scale ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Accurate Estimation ◽  
Content Type ◽  
Pooling Strategy ◽  
Stool Specimens

Shiga toxin-producingEscherichia coli(STEC)-associated enteric illness is attributed to O157 and non-O157 serotypes; however, traditional culture-based methods underdetect non-O157 STEC. Labor and cost of consumables are major barriers to implementation of the CDC recommendation to test all stools for both O157 and non-O157 serotypes. We evaluated the feasibility of a pooled nucleic acid amplification test (NAAT) as an approach for screening stool specimens for STEC. For retrospective evaluation, 300 stool specimens were used to create pools of 10 samples each. The sensitivity was 83% for the preenrichment pooling strategy and 100% for the postenrichment pooling strategy compared with those for individual NAAT results. The difference in cycle threshold (CT) between individual and pooled NAAT results for specimens was significantly lower and more consistent for postenrichment pooling (stx1mean = 3.90,stx2mean = 4.28) than those for preenrichment pooling (excluding undetected specimens;stx1mean = 9.34,stx2mean = 8.96) (P≤ 0.0013). Cost of consumables and labor time savings of 48 to 81% and 6 to 66%, respectively, were estimated for the testing of 90 specimens by the postenrichment pooled NAAT strategy on the basis of an expected 1 to 2% positivity rate. A 30-day prospective head-to-head clinical trial involving 512 specimens confirmed the sensitivity and labor savings associated with the postenrichment pooled NAAT strategy. The postenrichment pooled NAAT strategy described here is suitable for efficient large-scale surveillance of all STEC serotypes. Comprehensive detection of STEC will result in accurate estimation of STEC burden and, consequently, appropriate public health interventions.

scholarly journals Improved Genomic Identification, Clustering, and Serotyping of Shiga Toxin-Producing Escherichia coli Using Cluster/Serotype-Specific Gene Markers

2022 ◽  
Vol 11 ◽  
Author(s):  
Xiaomei Zhang ◽  
Michael Payne ◽  
Sandeep Kaur ◽  
Ruiting Lan
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
In Silico ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Specific Gene ◽  
Metagenomic Sequencing ◽  
Accurate Identification ◽  
Gene Markers ◽  
E Coli

Shiga toxin-producing Escherichia coli (STEC) have more than 470 serotypes. The well-known STEC O157:H7 serotype is a leading cause of STEC infections in humans. However, the incidence of non-O157:H7 STEC serotypes associated with foodborne outbreaks and human infections has increased in recent years. Current detection and serotyping assays are focusing on O157 and top six (“Big six”) non-O157 STEC serogroups. In this study, we performed phylogenetic analysis of nearly 41,000 publicly available STEC genomes representing 460 different STEC serotypes and identified 19 major and 229 minor STEC clusters. STEC cluster-specific gene markers were then identified through comparative genomic analysis. We further identified serotype-specific gene markers for the top 10 most frequent non-O157:H7 STEC serotypes. The cluster or serotype specific gene markers had 99.54% accuracy and more than 97.25% specificity when tested using 38,534 STEC and 14,216 non-STEC E. coli genomes, respectively. In addition, we developed a freely available in silico serotyping pipeline named STECFinder that combined these robust gene markers with established E. coli serotype specific O and H antigen genes and stx genes for accurate identification, cluster determination and serotyping of STEC. STECFinder can assign 99.85% and 99.83% of 38,534 STEC isolates to STEC clusters using assembled genomes and Illumina reads respectively and can simultaneously predict stx subtypes and STEC serotypes. Using shotgun metagenomic sequencing reads of STEC spiked food samples from a published study, we demonstrated that STECFinder can detect the spiked STEC serotypes, accurately. The cluster/serotype-specific gene markers could also be adapted for culture independent typing, facilitating rapid STEC typing. STECFinder is available as an installable package (https://github.com/LanLab/STECFinder) and will be useful for in silico STEC cluster identification and serotyping using genome data.

scholarly journals Prevalence and epidemiology of non-O157 Escherichia coli serogroups O26, O103, O111, O145 and Shiga toxin gene carriage in Scottish cattle, 2014-2015.

2021 ◽  
Author(s):  
Deborah V. Hoyle ◽  
Marianne Keith ◽  
Helen Williamson ◽  
Kareen Macleod ◽  
Heather Mathie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Clinical Situation ◽  
Herd Size ◽  
Seasonal Effects ◽  
Toxin Gene ◽  
Bacterial Strains ◽  
Herd Level ◽  
Contaminated Food ◽  
Serious Disease

Cattle are a reservoir for Shiga toxin-producing Escherichia coli (STEC), zoonotic pathogens that cause serious clinical disease. Scotland has a higher incidence of STEC infection in the human population than the European average. The aim of this study was to investigate the prevalence and epidemiology of non-O157 serogroups O26, O103, O111, O145, and Shiga toxin gene carriage, in Scottish cattle. Faecal samples (n = 2783) were collected from 110 herds between 2014-2015 and screened by real-time PCR. Herd-level prevalence (95% CI) for O103, O26 and O145 was estimated as 0.71 (0.62, 0.79), 0.43 (0.34, 0.52) and 0.23 (0.16, 0.32), respectively. Only two herds were positive for O111. Shiga toxin prevalence was high in both herds and pats, particularly for stx2 (herd-level: 0.99, 95% CI: 0.94, 1.0). O26 bacterial strains were isolated from 36 herds on culture. Fifteen herds yielded O26 stx-positive isolates that additionally harboured the intimin gene; six of these herds shed highly pathogenic stx2a-positive strains. Multiple serogroups were detected in herds and pats, with only 25 herds negative for all serogroups. Despite overlap in detection, regional and seasonal effects were observed. Higher herd prevalence for O26, O103 and stx1 occurred in the South West and this region was significant for stx2 at the pat-level (P = 0.015). Significant seasonal variation was observed for O145 prevalence, highest in autumn (P = 0.032). Negative herds were associated with Central Scotland and winter. Herds positive for all serogroups were associated with autumn, larger herd size and were not housed at sampling. IMPORTANCE Cattle are reservoirs for Shiga toxin Escherichia coli (STEC), bacteria shed in animal faeces. Human are infected through consumption of contaminated food or water, and by direct contact, causing serious disease and kidney failure in the most vulnerable. The contribution of non-O157 serogroups to STEC illness was underestimated for many years, due to the lack of specific tests. Recently non-O157 human cases have increased, with O26 STEC of particular note. It is therefore vital to investigate the level and composition of non-O157 in the cattle reservoir, and compare to historical levels and the clinical situation. In this study we found cattle prevalence high for toxin, as well as O103 and O26 serogroups. Pathogenic O26 STEC were isolated from 14% of study herds, with toxin subtypes similar to that seen in Scottish clinical cases. This study highlights the current risk to public health from non-O157 STEC in Scottish cattle.

scholarly journals Virulence Profiling of Shiga Toxin-Producing Escherichia coli O111:NM Isolates from Cattle

2013 ◽  
Vol 79 (13) ◽  
pp. 4164-4165 ◽  
Author(s):  
Musafiri Karama ◽  
Carlton L. Gyles
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Disease Outbreaks ◽  
The United States ◽  
Full Spectrum ◽  
Gene Markers ◽  
Content Type

ABSTRACTShiga toxin-producingEscherichia coli(STEC) O111:NM is an important serotype that has been incriminated in disease outbreaks in the United States. This study characterized cattle STEC O111:NM for virulence factors and markers by PCR. Major conclusions are that STEC O111:NM characterized in this study lacksstx2and the full spectrum ofnlegene markers, and it has an incomplete OI-122.

Pathogenic Escherichia coli in rural household container waters

2013 ◽  
Vol 67 (6) ◽  
pp. 1230-1237 ◽  
Author(s):  
P. Jagals ◽  
T. G. Barnard ◽  
M. M. Mokoena ◽  
N. Ashbolt ◽  
D. J. Roser
Keyword(s):  
Escherichia Coli ◽  
Water Quality ◽  
Rural Communities ◽  
Water Supply ◽  
Pcr Analysis ◽  
Gene Markers ◽  
E Coli ◽  
Bacterial Indicator ◽  
Domestic Use ◽  
Gene Data

Plastic containers in the range of 5–20 L are widely used – especially in rural African settings – to collect, transport and store water for domestic use, including drinking, bathing and hygiene. The pathogen content of the waters in these containers has not been adequately characterized as yet. This paper presents the primary findings of a synoptic survey of drinking water quality samples from these containers and involved collection of bacterial indicator and pathogenicity gene data. In total, 571 samples of a variety of waters were taken in rural communities in South Africa and the Escherichia coli numbers measured. Of the E. coli positive samples, 46% (n = 148) were screened for the presence of E. coli pathogen gene markers. Though synoptic, the survey provided many insights into the issues that drove the study. Container use markedly degraded water quality as judged by indicator counts, even where improved water supply services were in place. Household container use also appeared to promote regrowth or contamination of containers with pathogenic E. coli strains. Polymerase chain reaction (PCR) analysis also showed that the diversity of potential pathogenic E. coli carrying virulence genes was great. All seven genes screened for (Ial, Stx1, Stx2, EaeA, Eagg, ST, LT) were found in the waters, alone or as mixtures (number of different combinations = 31) including those characteristic of the more dangerous invasive and haemorrhagic E. coli strains. Given the central role of containers in the management of water supply to rural communities, it is clear the microbiology of these waters requires much further characterization.

Prevalence of Shiga toxin-producing and enteropathogenic Escherichia coli marker genes in diarrhoeic stools in a New Zealand catchment area

2016 ◽  
Vol 70 (1) ◽  
pp. 81-84 ◽  
Author(s):  
Rowan R Thomas ◽  
Heather J L Brooks ◽  
Rory O'Brien
Keyword(s):  
Escherichia Coli ◽  
New Zealand ◽  
Shiga Toxin ◽  
Marker Genes ◽  
Gene Markers ◽  
E Coli ◽  
Chelex 100 ◽  
Extraintestinal Disease ◽  
Somatic Antigens ◽  
Quantitative Pcr Assay

BackgroundShiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli are gastrointestinal pathogens causing diarrhoeal and extraintestinal disease. Due to lack of EPEC screening and use of Sorbitol-MacConkey (SMAC) agar in faecal screening, the true prevalence of EPEC and non-O157 STEC in New Zealand diarrhoeal cases is unknown.MethodsDiarrhoeic stools sourced from Dunedin hospital were pre-enriched, DNA extracted with Chelex-100 resin and screened using a multiplex TaqMan quantitative PCR assay amplifying stx1, sxt2 and EPEC (eae) gene markers.ResultsOf the 522 diarrhoeic samples surveyed, 8 (1.53%) were PCR positive for stx1/stx2 and 23 (4.41%) were positive for eae. Six (75%) of the stx+ samples were uncommon non-O157 serotypes, and the remainder were found to be positive for both O103 and O157 STEC somatic antigens.ConclusionsResults revealed shortcomings in current screening protocols for pathogenic E. coli; SMAC is not sufficiently discriminatory to detect emergent STEC serotypes and EPEC likely has an unappreciated role in cases of diarrhoea in New Zealand.

A Simple PCR-Based Macroarray System for Detection of Multiple Gene Markers in the Identification of Priority Enterohemorrhagic Escherichia coli

2011 ◽  
Vol 74 (3) ◽  
pp. 365-372 ◽  
Author(s):  
BURTON W. BLAIS ◽  
AMALIA MARTINEZ-PEREZ
Keyword(s):  
Escherichia Coli ◽  
Target Genes ◽  
Enrichment Culture ◽  
Enterohemorrhagic Escherichia Coli ◽  
High Rate ◽  
Antigenic Determinants ◽  
Bacterial Strains ◽  
Gene Markers ◽  
E Coli ◽  
Different Strains

Enterohemorrhagic Escherichia coli (EHEC) strains bearing the O antigenic determinants O157, O26, O111, O103, and O145 have a high rate of association with foodborne illness worldwide. To expand Canadian food inspection capability, a cloth-based hybridization array system (CHAS) was developed for the identification and characterization of priority EHEC. This method targets key virulence genes (eae, hlyA, vt1, and vt2) plus the rfbE gene specifying the O157 antigenic determinant, and the wzx genes specifying the O26, O111, O103, and O145 determinants. Multiplex PCR products incorporating a digoxigenin label were detected by hybridization with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. This method identified the relevant markers in 85 different strains bearing various combinations of the target genes (virulence and priority O-antigen markers). None of the target genes was detected in 26 different strains of other E. coli and non–E. coli bacteria. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the various markers among different bacterial strains. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the markers among various target and nontarget bacteria. The entire procedure could be completed in less than 5 h, and is useful for the identification of priority EHEC colonies isolated from foods by using enrichment culture techniques.

Multilaboratory Validation of a Luminex Microbead-Based Suspension Array for the Identification of the 11 Most Clinically Relevant Shiga Toxin–Producing Escherichia coli O Serogroups†

2013 ◽  
Vol 76 (5) ◽  
pp. 867-870 ◽  
Author(s):  
ANDREW LIN ◽  
JULIE A. KASE ◽  
MICHELLE M. MOORE ◽  
INSOOK SON ◽  
NELLY TRAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Shiga Toxin ◽  
False Positive ◽  
Collaborative Study ◽  
False Negative ◽  
Robust Method ◽  
Suspension Array ◽  
Negative Results ◽  
False Negative Results

Rapid and high-throughput identification and serotyping of Shiga toxin–producing Escherichia coli (STEC) O serogroups is important for detecting, investigating, and controlling STEC infection outbreaks and removing hazardous products from commerce. A Luminex microbead-based suspension array has been developed to identify the 11 most clinically relevant STEC serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. Here we present results of a blinded multilaboratory collaborative study involving 10 participants from nine laboratories using 55 unknown strains. From the total 495 analyses, two false-positive and three false-negative results were obtained, indicating the assay to be a rapid, high-throughput, and robust method for identifying clinically relevant STEC serogroups.

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